Bottom Line:
Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM.Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network.These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

ABSTRACTA novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Figure 7: Inhibition of microtubule nucleation by the anti-RanBPM antibodies. 0. 6 μl of the centrosome fraction were preincubated with 2.1 μg of the affinity-purified anti-RanBPM antibodies (+) or buffer alone (−) for 15 min at 37°C, and then tubulin was added to start the microtubule nucleation. The microtubule asters were spun down, fixed, and then double stained with affinity-purified anti-RanBPM antibodies (green) and the mAb to α-tubulin (red). Both staining patterns were superimposed by electronic image processing (superimposition). Bars, 5 μm.

Mentions:
To determine the localization of RanBPM in the microtubule asters assembled in vitro, the asters were doubly stained by the mAb to α-tubulin (green) and by the affinity-purified anti-RanBPM antibodies (red). As expected from the in vivo results, RanBPM was localized at the central part of the microtubule asters (Fig. 6) where γ-tubulin was also localized (see Fig. 9). These results are consistent with the notion that RanBPM is one of the centrosomal components which is involved in microtubule nucleation. To address this issue, the centrosome fractions were preincubated with the affinity-purified anti-RanBPM antibodies, and then assayed for aster formation. Although the preimmune IgG had no effect on aster formation, the number of microtubules nucleated by the centrosome was greatly reduced by addition of anti-RanBPM antibodies (Fig. 7). In a control reaction mixture containing the preimmune IgG or the buffer alone, the total number of asters formed on a coverslip was 1442 (SD = 27.0) and 1489 (SD = 51.1), respectively. In contrast, it was 151 (SD = 10.1) in the presence of anti-RanBPM antibodies. Thus, the aster-forming ability of the centrosome factions was reduced to about 10% of the buffer alone by the addition of the affinity-purified anti-RanBPM antibodies. Under the same conditions, the polymerization of microtubules without centrosome fractions was not inhibited by the affinity-purified anti-RanBPM antibodies (data not shown), this being consistent with the result showing that the length of the microtubules was not reduced by the addition of anti-RanBPM antibodies (Fig. 7).

Figure 7: Inhibition of microtubule nucleation by the anti-RanBPM antibodies. 0. 6 μl of the centrosome fraction were preincubated with 2.1 μg of the affinity-purified anti-RanBPM antibodies (+) or buffer alone (−) for 15 min at 37°C, and then tubulin was added to start the microtubule nucleation. The microtubule asters were spun down, fixed, and then double stained with affinity-purified anti-RanBPM antibodies (green) and the mAb to α-tubulin (red). Both staining patterns were superimposed by electronic image processing (superimposition). Bars, 5 μm.

Mentions:
To determine the localization of RanBPM in the microtubule asters assembled in vitro, the asters were doubly stained by the mAb to α-tubulin (green) and by the affinity-purified anti-RanBPM antibodies (red). As expected from the in vivo results, RanBPM was localized at the central part of the microtubule asters (Fig. 6) where γ-tubulin was also localized (see Fig. 9). These results are consistent with the notion that RanBPM is one of the centrosomal components which is involved in microtubule nucleation. To address this issue, the centrosome fractions were preincubated with the affinity-purified anti-RanBPM antibodies, and then assayed for aster formation. Although the preimmune IgG had no effect on aster formation, the number of microtubules nucleated by the centrosome was greatly reduced by addition of anti-RanBPM antibodies (Fig. 7). In a control reaction mixture containing the preimmune IgG or the buffer alone, the total number of asters formed on a coverslip was 1442 (SD = 27.0) and 1489 (SD = 51.1), respectively. In contrast, it was 151 (SD = 10.1) in the presence of anti-RanBPM antibodies. Thus, the aster-forming ability of the centrosome factions was reduced to about 10% of the buffer alone by the addition of the affinity-purified anti-RanBPM antibodies. Under the same conditions, the polymerization of microtubules without centrosome fractions was not inhibited by the affinity-purified anti-RanBPM antibodies (data not shown), this being consistent with the result showing that the length of the microtubules was not reduced by the addition of anti-RanBPM antibodies (Fig. 7).

Bottom Line:
Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM.Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network.These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

ABSTRACTA novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.